This application is related to the co-pending application entitled METHOD OF PERFORMING RADIOIMMUNOASSAY AND KIT, Ser. No. 08/581,250, filed, and owned by the same inventor.
This invention relates generally to medical diagnostics and, more particularly to a simplified method of preparing and activating protein materials in a biologic sample, for example human serum, for radioimmunoassay or other diagnostic tests.
The test of the present invention is based on the discovery of a novel gene, denoted METALLOPANSTIMULIN (MPS), which encodes a protein associated with many human cancers. The novel DNA sequence which encodes the METALLOPANSTIMULIN is described in detail in U.S. Pat. No. 5,243,041, which is hereby incorporated by reference. METALLOPANSTIMULIN is "zinc finger" protein of subunit molecular weight approximately 10,000-Dalton is designated as "METALLOPANSTIMULIN" (MPS) since:
1) it forms a complex with metal ions such as zinc (Greek: metallo); PA1 2) it has been detected in many different cell types (Greek: pan=all); PA1 3) it is associated with ribosomes; and PA1 4) it is associated with rapid cell proliferation (Latin: stimulin). PA1 1) it has a nuclear localization signal and it can bind specific DNA sequences probably acting as a modulator of transcription; PA1 2) the MPS protein stimulates cell growth; and PA1 3) the MPS protein is secreted from the cells into extracellular fluids. PA1 1) detection of primary disease in asymptomatic, previously undiagnosed individuals; and PA1 2) detection of cancer recurrence in previously diagnosed and treated patients. PA1 1) Detection of primary disease in previously undiagnosed individuals; and PA1 2) Detection of cancer recurrence in previously diagnosed and treated patients.
MPS is a multifunctional protein with at least three main functions:
Numerous experiments with human tissue culture cells and human pathological tissue specimens demonstrated that the MPS-1 mRNA and its encoded protein are expressed in normal cells to a much lesser degree than in premalignant or malignant tumor cells, and they are present at very low levels in senescent cells compared to young healthy cells.
It is believed that cancer detected early is more susceptible to therapy than cancer detected late.
Early detection of cancer is important in two general situations:
The ability to identify cancer at an early stage may be useful to administer an effective therapy.
A reliable test for detecting malignant cell proliferation, when the tumor is confined and effective treatment can be provided, would be of great value for the physician. At present, the majority of cancers have advanced beyond the organ of origin at the time of diagnosis.
The measurement of MPS-N and MPS-N-like materials in human serum is of primary interest in the detection of active malignant proliferative processes. For example, the results of clinical correlation studies to date indicate that MPS-N is frequently (&gt;90% of the cases) elevated (&gt;10 ng/mL) in a large number of active common malignancies, such as prostate, colorectal, lung and other cancers. Thus, MPS-N testing has significant value in detecting undiagnosed malignant cell proliferation and/or active oncogenic processes. The monitoring of patients with diagnosed malignancies in which changes in concentrations of MPS-N are observed is also of significant clinical value.
An extensive clinical study including 632 individuals separated in healthy subjects, active cancerous diseases, non-malignant diseases, and premalignant diseases has provided important information about the use of MPS-N in the detection of various types of cancer. The data from the clinical study is provided below in Table 1 and also illustrated graphically in FIGS. 1A and 1B.
In general, the MPS-N test was found to be useful in:
TABLE 1 __________________________________________________________________________ DISTRIBUTION OF MPS VALUES NUMBER OF PATIENTS: 632 Percent (ng/mL) Number &lt;0.7 7.0-10 10.01-20 20.01-50 &gt;50.01 __________________________________________________________________________ HEALTHY SUBJECTS Women (19-64 years) 20 70 20 10 0 0 Men (21-55 years) 20 65 10 25 0 0 Men (50-88 years) 107 62 20 18 0 0 TOTAL 147 64 18 17 0 0 CANCEROUS DISEASES, ACTIVE Genitourinary Tract Prostate 126 1 &lt;1 11 48 38 Bladder 6 0 0 0 33 66 Testicular 1 0 0 0 0 100 Gastrointestinal Tract Esophageal 3 0 0 33 66 0 Pancreatic 1 0 0 0 0 100 Hepatoma 2 0 0 0 50 50 Colorectal 27 0 0 7 44 48 Lung Cancer Epithelial malignancies 27 0 0 11 26 63 Head and Neck Region Epithelial malignancies 6 0 0 0 66 33 Central Nervous System Primary Neoplasms 1 0 0 0 0 100 Neuroendocrine origin 6 0 0 17 17 66 Leukemia and lymphoma 7 0 0 0 43 57 Other Malignancies* 12 0 0 0 58 42 TOTAL 225 &lt;1 &lt;1 9 45 44 NONMALIGNANT DISEASES Benign Prostatic Hypertrophy 37 30 16 38 13 3 Hepatitis, B,C. 18 83 5 11 0 0 Liver cirrhosis 4 100 0 0 0 0 Other 201 58 21 20 1 0 TOTAL 260 56 19 21 3 &lt;1 Premalignant disease Colorectal Polyps 4 0 0 0 75 25 __________________________________________________________________________ *Other Malignancies: include cancer of unknown origins, squamous cell carcinomas, etc.
As illustrated, the quality criteria of the MPS-N test as a tumor marker, characterized by its diagnostic specificity (percentage of healthy individuals [82%] or benign diseases [75%] with a true negative test result); its sensitivity (percentage of true positive test results if cancer disease is present [98%]); and the cutoff value (concentration of MPS-N that differentiates healthy subjects or patients with benign disorders from those with premalignant or malignant disorders) clearly indicates that the MPS-N test has high specificity (&gt;75%) and sensitivity (&gt;98%) as a tumor marker.
The quantity of the MPS-N marker detectable is proportional to the extent of active malignancy as demonstrated by the following example of patients with prostatic carcinoma (PC). In patients not having PC, the MPS-N levels were lower than 10 ng/mL. In untreated patients having PC stages T1/T2, the MPS-N level range was 10-30 ng/mL; in stages T3/T4 the MPS-N level range was 30-50 ng/mL; and in stage M1b (distant metastasis to bone) the MPS-N levels were extremely high (100-500 ng/mL). In M1b patients that did not respond to therapy, the MPS-N levels remained very high (200 ng/mL). In M1b patients that went into remission after treatment, the MPS-N levels were greatly reduced. Thus, the increase in serum MPS-N correlated with the stage of the disease and response to therapy. The foregoing data originally was published in Fernandez-Pol, J. A, Klos, D. J., and Hamilton, P. D., The Evaluation of Metallopanstimulin As A Novel Tumor Marker in Sera of Patients With Prostatic Carcinoma, European J. of Nuclear Medicine, Supp. Vol. 21, No. 10; 94 (Abs)(1994); Fernandez-Pol, J. A., Klos, D. J., and Hamilton, P. D., Metallopanstimulin As A Novel Tumor Marker In Sera Of Patients With Prostatic Carcinoma, J. Tumor Marker Oncology, Supp. 1, June 1995 (Abs. No. A-49) p 54, which are hereby incorporated by reference.
Therefore, detection of primary prostate cancer in previously undiagnosed individuals can be accomplished by the serial measurement of MPS-N, provided that other types of cancers producing MPS-N materials are excluded. When employed for the management of prostate cancer patients, serial measurements of MPS-N is useful in detecting residual tumor and recurrent cancer after radical prostatectomy. Moreover, MPS-N is useful for assessing response of prostate cancer to therapy by prostatectomy, radiotherapy, and hormone therapy. Thus, serial measurements of MPS-N concentrations can be an important indicator in monitoring patients with prostate cancer and in determining the actual effectiveness of surgery and/or other treatments.
The MPS-N test also has significant importance in the detection of numerous types of undiagnosed common malignancies. As shown in Table 1, increased MPS-N levels have been detected with high frequency (&gt;90% of the cases) in numerous types of common cancers such as prostate, colorectal, lung, neuroendocrine, leukemias, etc. Moreover, in patients having these types of cancers, MPS-N testing can have important value in monitoring metastatic or persistently active cancer, following chemotherapy, surgery, or radiotherapy. A persistent elevation in circulating MPS-N levels following treatment or increase in an otherwise lower level is indicative of recurrent or residual cancer and poor therapeutic response. A declining MPS-N value is generally indicative of a good response to treatment and a favorable prognosis.
Serum MPS-N can also be elevated in active nonmalignant tumorigenic processes and premalignant proliferative conditions such as benign prostatic hypertrophy and colorectal polyps, respectively (Table 1). Inflammatory conditions of the prostate, liver, intestine and colon are negative for MPS-N (Table 1, section on "Hepatitis, Cirrhosis, Other").
It has been shown that the MPS-1 DNA sequence and the protein can be used in diagnostic methods such as detection of malignant cells associated with several types of tumors. The development of a sensitive and specific radioimmunoassay (RIA) for MPS-N, using synthetic peptide technology, would make it possible to detect the very low concentrations of MPS-N and MPS-N-like materials in human blood and other body fluids. Thus, the MPS-N RIA provides a method for determining the presence of certain types of abnormal proliferative conditions and/or active oncogenic processes in patients as described below.
Although it was suggested in the '041 patent that radioimmunoassay techniques possibly could be employed to detect MPS levels, a simple and cost effective method for performing radioimmunoassay on a large number of individual samples, for example in a routine clinical screening, was not taught or disclosed. Furthermore, no reference known to the inventor teaches or suggests a low-cost kit containing materials that can easily and economically be used by a diagnostician to perform radioimmunoassay for MPS-N like materials.
More importantly, no prior art reference suggested or taught a method of activating the MPS-like proteins in the serum sample which would allow detection of the MPS-N antigen. Most intracellular proteins that are released into the circulation are carried by binding proteins such as albumin, prealbumin and other more specific carrier proteins. It is well known that antibodies developed against peptide sequences of a given protein will not react in a radioimmunoassay system against native protein because most of the epitope (antigenic determinant that interact with the specific antibody) is masked. Intracellular proteins such as MPS are amphipatic and are dimeric or tetrameric in their native, non-denatured form. Under these conditions, certain regions of the polypeptide, such as epitopes corresponding to the N-terminal region, are unavailable for interaction with antibodies. Thus, the detection of the protein by antibodies is practically impossible. For example, only small quantities (2-10 ng/mL) of MPS can be detected in the native state in patients with metastatic cancer. The detectable concentration most likely corresponds to the free, unbound monomeric form of MPS and MPS-like materials.
A number of methods could be used to release, denature, linearize and, therefore, unmask or activate the total MPS-N and MPS-N-like protein. For example, acidification with 1N hydrochloric acid will release the protein. However, to measure the MPS-N a subsequent step of neutralization with sodium hydroxide is required. Also, a denaturing chaotropic agent to linearize the protein is required, with further extraction, including an expensive and cumbersome dialysis procedure. Some of the agents employed in these methods will activate proteases which will, in turn, digest the protein of interest. These are sensitive steps that may lead to inaccurate results upon measurement of the protein. The method of using chemical activation with hydrochloric acid, neutalization with sodium hydroxide plus extraction with solvents in employed in the immunoassay test for measurement of Transforming Growth Factor Beta in serum. These procedures are time consuming and subject to error making it quite difficult to make a precise determination of MPS.